Pneumonia caused by Pneumocystis (PcP) remains a major opportunistic infection associated with AIDS patients, even in the era of Highly Active Anti-Retroviral Therapy (HAART) (Castro, 1998; Centers for Disease Control and Prevention, 1999). In the previous 2 decades, patients with AIDS have been a primary target of PcP, the population in which it remains a leading opportunistic infection. Limited therapeutic choices and adverse reactions to the two standard treatments, trimethoprim-sulfamethoxazole (TMP-SMX) and pentamidine (Mei, Gurunathan et al., 1998), cause the clinical management of this infection to remain problematic. Moreover, side effects in almost half of AIDs patients required switching to a less effective therapy (Wilkin & Feinberg, 1999). Despite the efforts of several in vitro and in vivo screening projects, no better treatment than TMP-SMX for PcP has been identified (Walzer, Foy et al., 1992b; Wilkin & Feinberg, 1999). Strategies to exploit the effective combination of dihydrofolate reductase inhibitor and DHPS inhibitor of the TMP-SMX combination, by substitution of each component (e.g. TMP-dapsone) have not resulted in any therapies with increased efficacy. More recently, mutations in Pneumocystis genes which are the targets of TMP-SMX ((Ma, Borio et al., 1999; Mei, Gurunathan, Masur, & Kovacs, 1998)), atovaquone (Walker, Wakefield et al., 1998) and dapsone (Kazanjian, Armstrong et al.; Kazanjian, Armstrong et al.) were similar to those conferring resistance in other organisms such as Plasmodium falciparum. Previous therapy with these agents had a strong correlation to presence of the mutation, suggesting a selective mechanism was operational. Moreover, a Pc genotype with double mutations in the DHPS gene replaced the wild type genotype (no mutations) as the predominant type in certain regions of the country (e.g. San Francisco) implying that again, a dominant selection was occurring and these organisms were being transmitted throughout the human population in these regions (Beard, Carter et al.).
The limited repertoire, problems in tolerance, and potential emerging resistance make it necessary to identify new efficacious treatments for PcP. Drug screening and development for anti-PcP agents has taken advantage of available rodent models of PcP and short term in vitro systems. Recombinant proteins have been used in some biochemical assays when the Pc gene was cloned as in the case of dihydrofolate reductase, but this application has been rarely used due to the paucity of Pc gene sequences previously available.
The rodent model of PcP was developed in 1966 (Frenkel, Good et al., 1966) and since then has been used for the general study of these organisms and for drug development. The efficacy of a compound is evaluated by the decrease in organism numbers in the lungs of treated versus non-treated rats or mice. Organisms can be quantified by microscopic counting methods or by estimation of organism burden using a semi-quantitative scoring system.
An ATP assay system (Cushion, Chen et al., 1997; Chen & Cushion, 1994; Collins & Cushion, 2001) was used to evaluate bisbenzamidine analogues as potential therapeutic targets for treatment of pneumonia, particularly that caused by species in the genus Pneumocystis. These compounds exhibited activity against Pneumocystis with little or no toxicity, in vitro. Development of these compounds could provide new and novel treatment choices for Pneumocystis pneumonia.
The ATP system was used to evaluate the activity of pentamidine analogues and to evaluate their toxicity to three human derived cell lines, A549, Hep-G2 and WI-38.